Question

I am learning RNA-seq analysis. I always encounter this phase "poly(A)+ RNA". After searching, I got this: "Most messenger RNAs contain a poly(A) tail, while structural RNAs do not. Poly(A) selection therefore enriches for messenger RNA. The technique has proved essential for construction of cDNA libraries."

Does it mean that when constructing the cDNA, we always use the ones with a A's tail? Then other RNAs like tRNA are filtered and not used to get their cDNA? I do find some RNAs records on UCSC where microRNAs are included.

When we say poly(A)+ RNA library, what is the difference between normal one? Or normal RNA library is a poly(A)+ RNA library?

Answer 1

One of the primary reasons to use poly(A) selection is to eliminate the massive amount of ribosomal RNA (rRNA) present in the samples. The alternative is to use ribosomal RNA depletion kits / techniques to remove as much rRNA as possible before sequencing. Without rRNA depletion a large proportion (~60-80%) of the reads would map to rRNAs.

poly(A) selection also reduces the amount of pre-mRNA in the samples, which reduces the number of intron aligned reads.

Answer 2

The RNA library includes all types of RNA like mRNA, rRNA, tRNA,snRNA. all the different types of RNA cannot be used for cDNA synthesis. Hence, poly-A tail of mRNA differentiates it from other RNA types.

The cDNA is the double stranded DNA that is synthesized from mRNA by using an enzyme called reverse transcriptase. The poly- A tail of mRNA also differentiates pre mRNA from matured mRNA. The matured mRNA undergoes translation to synthesize protein along with rRNA and tRNA.

The poly-T tail is used in cDNA synthesis which base pairs with poly-A tail of mRNA. Thus, obtained matured DNA is reverse transcribed to form cDNA.

The cDNA is used in gene cloning. It is also used as gene probe or for constructing cDNA libraries.